Nitrogen assimilation is of fundamental importance to the growth of plants. Of all the mineral nutrients required by plants, nitrogen is required in the greatest abundance. The main forms of nitrogen taken up by plants in the field are nitrate and ammonia, the principle components of nitrogenous fertilizers. Plants take up either nitrate or ammonium ions from the soil, depending on availability. Nitrate will be more abundant in well-oxygenated, non-acidic soils, whilst ammonium will predominate in acidic or water-logged soils. Experiments on growth parameters of tobacco clearly demonstrated that relative growth rate, chlorophyll content, leaf area and root area increased dramatically in response to increasing nitrate supply.
Plants have developed an efficient nitrogen uptake system in order to cope with the large variation in nitrate content of cultivated soils. Plant roots take up nitrate and ammonia by the action of specific nitrate transporters (NTR), which are divided into two gene families, the NRT1 gene family and the NRT2 gene family. It is generally assumed that the NRT1 gene family mediates the low-affinity nitrate transport system (LATS, i.e. when the external nitrate concentration >1 mM), with the exception of the AtNRT1.1, which is both a dual affinity transporter and a nitrate sensor, while the NRT2 gene family mediates the high-affinity nitrate transport system (HATS, i.e. when the external nitrate concentration <1 mM). In Arabidopsis, 53 genes belong to the NRT1 family, however, only 51 of these genes are transcribed and each within various tissues. This implies that the polypeptides that they encode possess specialized or unique functions. For example, Atnrtl.2 is constitutively expressed, but only within the root epidermis of Arabidopsis, where it participates in the constitutive low-affinity nitrate transport system.
Once within the roots of a plant, nitrate is then loaded into the xylem of the vascular stele where it is then distributed to other organs within the plant. It is known that the low affinity nitrate transporter, NRT1.5, plays a major role in loading nitrate, which is present within the roots, into xylem vessels. Once inside a cell, nitrate can be exported from vacuoles and transported towards growing organs, such as young leaves, when the availability of external supplies is limited or when mature leaves age and become a source leaves (i.e. leaves that pass on their nutrients to other tissues within a plant).
Nitrate remobilization is responsible for recycling nitrogen present within older leaves. It passes recycled nitrate onto younger leaves in order to sustain the growth of developing tissues. Nitrate remobilization also occurs between leaves and seeds, and is important for grain production and nitrogen recycling by senescent leaves. It is estimated that the contribution of leaf nitrogen remobilization to grain production is cultivar dependent, varying from 50 to 90%. Remobilized nitrogen may be in the form of amino acids released by proteolytic activity. However, inorganic nitrogen, in the form of nitrate, may also be remobilized. Thus, membrane proteins must transport nitrate through the plasma membrane towards the vascular tissues of source organs.
Nitrate within source organs is then transported across the plasma membrane into the mesophyll cells. Here it is either stored in vacuoles, or reduced in the cytoplasm and enters the primary nitrogen assimilation pathway. When nitrate is present in excess, it is stored in the vacuole. This serves both as an osmoticum (i.e. supplements osmotic pressure), and as a source of mineral nitrogen to be used when nitrate uptake is minimal. The nitrate present in the cytoplasm is the starting point of primary nitrogen assimilation.
Nitrate is reduced in the cytosol by the cytoplasmic enzyme nitrate reductase to nitrite, which itself is rapidly reduced to ammonium by nitrite reductase (NiR) in the chloroplasts of leaves or in the plastids of non-photosynthetic organs. In the chloroplast, the ammonium then enters the glutamine synthetase/glutamate synthase cycle (GS/GOGAT), where it is incorporated into the amino acid pool.
The regulation of the activities of nitrate transporters, and nitrate and nitrite reductases is critical in controlling primary nitrogen assimilation throughout the plant, and has a significant impact on the growth and development of the plant. However, under certain conditions, nitrate may accumulate, mainly in green photosynthetically active tissues, where it is stored in the vacuoles of the mesophyll cells. High levels of nitrate accumulation can occur during periods of low temperature and/or solar irradiation (for example, in greenhouse crops during the winter), when there is less photosynthetic capacity to assimilate the stored nitrate, or as a result of high nitrate levels in the soil. An increase in nitrate levels can have a number of deleterious consequences, not only in terms of plant growth, but also in terms of human or animal health where the plant is consumed, as well as environmental consequences. Many of the adverse consequences of nitrate accumulation are mediated through the production of nitrite.
Therefore, to prevent nitrate accumulation, one strategy would be increasing nitrogen remobilisation in plants, for example when they become senescent, which could have important applications in crop production. Firstly, nitrogen remobilised from leaves can be transported to the younger leaves as well as the developing seed. Increasing the efficiency of nitrogen exit from senescent leaves could therefore potentially increase nitrogen supply to seeds and younger parts of the plant, and thereby increase crop yield and nitrogen use efficiency. This is clearly a valuable goal when the world population is increasing but crop yields are not increasing sufficiently to meet demand. One potential target crop is Brassica napus (oilseed rape), which has poor nitrogen efficiency due to poor nitrogen remobilisation from vegetative tissue. Another target crop is wheat, as the potential benefits of increasing grain protein content are great. Grain protein content not only affects nutritive value of wheat, but also determines grain usage and therefore market value. For example, increased grain protein content results in increased bread volume.
Also, an ability to increase nitrogen remobilisation could be very useful in the tobacco industry because it is known that residual nitrogen in tobacco leaves contributes to the formation of nitrosamines, as illustrated in FIG. 1. In particular, nitrate and nitrite act as precursors to tobacco-specific nitrosamine (TSNA) formation in cured leaf. The processing of the tobacco leaves by the tobacco industry involves the removal of petioles and mid-ribs of the cured leaves which are believed to act as nitrate storage organs, devoid of flavour and high in TSNAs. Also, the formation of nitrosamines in the stomach is a result of endogenous nitrosation. Oral bacteria chemically reduce nitrate consumed in food and drink to nitrite, which can form nitrosating agents in the acidic environment of the stomach. These react with amines to produce nitrosamines and cause DNA strand breaks or cross linking of DNA. Another problem associated with an excess of nitrate is the formation of methaemoglobin which gives rise to blue baby syndrome, where the oxygen carrying capacity of haemoglobin is blocked by nitrite, causing chemical asphyxiation in infants.
As a consequence of these health concerns, a number of regulatory authorities have set limits on the amount of nitrate allowed in leafy green vegetables such as spinach and lettuce (e.g. European Commission Regulation 653/2003), depending on the time of harvest. These limits have resulted in any produce with a high nitrate content being unmarketable. Consequently, there have been efforts to reduce nitrate content of plants by managing the application of nitrogen-containing fertilisers or improved systems of crop husbandry. Some authorities have also set limits on the amounts of nitrate in drinking water.
There is therefore a need for means for alleviating the adverse effects associated with nitrate accumulation in plants. With this in mind, the inventors have developed a series of genetic constructs, which may be used in the preparation of transgenic plants, which exhibit surprisingly reduced nitrate concentrations.